Acquired immune deficiency syndrome (AIDS) was first recognized in approximately 1981, but the causative agent had not yet been identified. Intense research efforts resulted in the detection and isolation of HTLV-III, the retrovirus identified as the etiologic agent of AIDS.
The virus is currently believed to be transmitted through intimate sexual contact and blood. Thus far, epidemiologic evidence indicates that food, water, insects and casual contact are not disease vectors.
The AIDS virus acts by crippling the body's immune system. Particularly, HTLV-III selectively attacks T.sub.4 lymphocytes, one of the subpopulations of lymphocytes that constitute the immune system. Infection with HTLV-III results in both a reduction in the number and a change in the function of the targeted T.sub.4 lymphocytes with the eventual collapse of the immune system.
The groups at highest risk of infection with HTLV-III include homosexual and bisexual men and abusers of injected drugs. Other predictable high risk groups are women artificially inseminated with sperm from infected donors and sexual partners of those in the AIDS risk groups. Recipients of blood transfusions and blood products are also at risk of contracting AIDS.
Organ transplant recipients are also a high risk group. Recognizing organ transplantation as a potential avenue for the spread of AIDS, testing for HTLV-III antibodies is now performed on all organ donors. With the exception of some kidney transplants it is often difficult to predict in advance the source of a donated organ. For example, victims of fatal automobile accidents are potential organ donors, and as the time and place of accidents are not foreseeable, pre-transplant testing presents a problem. Screening is thus difficult in this and similar situations and the need for 24 hour testing facilities and, more importantly, on site testing is apparent. Further, in view of the limited useful life of a donated organ once removed from the host, the need for a rapid assay method is evident.
Present methods for the detection of antibodies to the AIDS virus include the enzyme-linked immunosorbance assay (ELISA), Western Blot and immunofluorescent techniques. Such assays are described in Sarngadharan, M.G. et al., Science 224:506 (1983) (ELISA); Tsang et al., In: Methods of Enzymology, Vol. 92, Chapter 29, 1983, Academic Press and Safai, B. et al., Lancet 1:1438 (1984) (Western Blot), and Essex, M. et al., Science 220:859 (1983) (immunofluorescent techniques). The established method for blood donor screening is to first carry out an ELISA, followed by confirmation of positives by Western Blot.
The ELISA technique involves reacting a test sample with an antigen reagent generally obtained from disrupted whole or density-banded HTLV-III. Typically, the HTLV-III is coated onto wells of a microtiter plate. After washing to remove unbound antibodies, goat-antihuman IgG antiserum conjugated with horseradish peroxidase is added to the wells and incubated. After an appropriate incubation period, an enzyme substrate is added to the mixture and a detectable, measurable color product is formed in the presence of antibodies to HTLV-III.
In the Western Blot technique, on the other hand, the HTLV-III antigen is electrophoretically resolved on SDS-polyacrylamide gels, each 8.times.10 cm gel being loaded with5-10 .mu.g of protein. The resulting protein bands are electro-transferred to nitrocellulose paper. Detection of antibodies to HTLV-III is then carried out by either solid phase strip radioimmunoassay techniques or by ELISA. Each of these methods includes an overnight incubation, and thus, an overall test time of about 20 hours.
The established screening procedure is not entirely satisfactory because of the time required to obtain results. This is particularly true in the organ transplant situation, especially for the heart and liver. These two organs have maximum cold ischemic times of 4 and 8 hours, respectively. Generally, ELISA takes about 4 hours and the Western Blot which, as noted, includes an overnight incubation period, requires about 20 hours. As can be appreciated, with conventional techniques test results would not be obtained within the maximum ischemic times for the heart or liver.
Accordingly, it is an object of the present invention to provide a rapid and sensitive assay for the detection of antibodies to HTLV-III whereby test results are obtained in under two hours, preferably before organ procurement is completed, but at least within the ischemic periods of the organs to be transplanted. It is a further object to provide diagnositc test kits for performing the assay method of the invention. Further objects and advantages of the present invention will become apparent upon reading the following description.